Selenium occurs as an integral component of several proteins in both prokaryotes and eukaryotes, usually as a selenocysteine moiety. The selenium in this amino acid is derived from a reactive selenium donor, selenophosphate. Escherichia coli selenophosphate synthetase catalyzes the formation of selenophosphate, AMP, and orthophosphate from selenide and ATP in a 1:1:1 ratio. While the enzyme mechanism was believed to proceed through an enzyme-pyrophosphoryl intermediate, experiments designed to prove this have been inconclusive. To help elucidate the mechanism, the enzyme was extensively purified for use in isotope studies with labeled ATP, which were carried out in the laboratory of Frank Raushel at Texas A&M University. The results of these experiments indicate that there is an enzyme-catalyzed cleavage at the g-phosphoryl of ATP to form an enzyme-phosphoryl intermediate. To obtain further evidence for an enzyme-phosphoryl intermediate, isotope exchange studies were carried out in the absence of selenide to determine if exchange between radioactive ADP and unlabeled ATP could be detected. These experiments showed that a slow exchange between ADP and ATP is catalyzed by the enzyme in the absence of selenide, with 11 percent of the radioactivity being recovered in the ATP. Furthermore, a significant amount of AMP, 32 percent of the recovered radioactivity, was also produced. When ADP was used as the only substrate, AMP was produced at a rate similar to that obtained in the exchange experiments. To determine if the slow rates of exchange are due to inefficient binding of ADP, experiments are currently being carried out to determine the Km for ADP. The enzymatic hydrolysis of ATP was also studied using phosphorous-NMR. In these experiments, the enzyme reaction was carried out in oxygen-18 water. Only the orthophosphate product contains oxygen-18. Our results thus far indicate that ADP is an intermediate in the reaction mechanism. Studies to further characterize the mechanism of action of this protein are being continued.